EXTENDED SPECTRUM BETA (Β) LACTAMASES IN CLINICAL ISOLATES OF GRAM-NEGATIVE BACILLI IN AJMAN, UNITED ARAB EMIRATES

GMJ ORIGINAL ARTICLE EXTENDED SPECTRUM BETA (Β) LACTAMASES IN CLINICAL ISOLATES OF GRAM-NEGATIVE BACILLI IN AJMAN, UNITED ARAB EMIRATES Ekta Chourasia, Krishna Pratap Singh, Sudheer Keshav Kher ABSTRACT Objective: Extended spectrum beta lactamase (ESBLs) producing strains of Enterobacteriaceae and Pseudomonas have emerged as a major problem worldwide. ESBLs, a major group of beta lactamases, are being identified in large numbers along with inducible AmpC beta lactamases and derepressed mutants. Many clinical laboratories currently test Gram-negative bacilli for ESBL production. However, there is scarcity of information from United Arab Emirates (UAE). This study was conducted to estimate the frequency of ESBLs and to detect different mechanisms of beta lactam resistance (ESBLs, AmpC beta lactamases, derepressed mutants, multiple mechanisms) in Gram-negative bacilli in Ajman, UAE. Materials & Methods: A total of 123 isolates of Gram-negative bacilli were tested for ESBL production. Screening and confirmatory tests for ESBL production were done according to Clinical Laboratory Standard Institute s guidelines. The novel method by Rodrigues et al was used to detect different mechanisms of resistance. Results: Forty-eight (39.1%) isolates showed ESBL production, of which 12(9.8%) were plain ESBL producers and multiple mechanism was exhibited by 36(29.3%). Nine(7.3%) were plain derepressed mutants. Inducible AmpC beta lactamase production was detected only in Pseudomonas aeruginosa(3.3%). The most common ESBL producer was E.coli (60.4%), however Klebsiella was the most frequent ESBL producer(40%). Forty one percent of ESBL producers were isolated from urine. All the isolates were sensitive to Imipenem. Conclusion: The occurrence of high frequency of ESBL-producing Gram-negative bacilli in our hospital is alarming and urgent action required to be taken for screening and confirmation of ESBL production along with continued surveillance, judicious use of antimicrobial agents as well as implementation of infection control measures. Keywords: Beta-lactam resistance, ESBL, Gram-negative bacilli, Enterobacteriaceae, UAE Citation: Chourasia E, Singh KP, Kher SK. Extended spectrum beta (β) lactamases in clinical isolates of gram-negative bacilli in Ajman, United Arab Emirates. Gulf Medical Journal. 2015;4(1):14-21. INTRODUCTION β-lactam antibiotics are one of the most frequently prescribed antimicrobial agents worldwide. The emergence of resistance to these antibiotics in the past few decades has resulted in a major clinical crisis 1,2. The most common cause of bacterial resistance to β-lactam antibiotics is the production of β-lactamase enzymes. Most of the second and third generation penicillins and cephalosporins were specifically designed to resist the hydrolytic action of major β-lactamases. Unfortunately, the battle between antibiotics and bacteria possessing β-lactamase, is far from over. As new classes of β-lactam antibiotics Correspondence: Dr. Ekta Chourasia, 80 Jellicoe Road, #07-01, Singapore 208766 Email: ektachourasia@gmail.com, Contact no 006584023413. are introduced, bacteria develop novel ways of overcoming these antibiotics 3. The indiscriminate use of newer antibiotics in the treatment of patients has given an edge to the new variants of β-lactamases, and the major group forming variant is extended spectrum β-lactamase (ESBL). In last few years, there has been an increased incidence and prevalence of ESBLs, enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam 4,5. It is unclear whether we will be able to keep pace with bacterial genetic changes and be able to effectively treat Gram- negative infections in future. ESBLs are currently being identified worldwide in large numbers and a significant percentage has been seen in strains of E.coli and K.pneumoniae. They have also been found in Pseudomonas aeruginosa, other Enterobacteriacae strains GMJ. NOVEMBER 2015: VOLUME 4(1). 14

EKTA CHOURASIA, ET AL like Enterobacter, Citrobacter, Proteus, Morganella morganii, Serratia marsescens, Shigella dysenteriae and non- Enterobacteriaceace Gram-negative bacilli 4,5. Production of these enzymes is either chromosomally mediated or plasmid mediated 5,6. Point amino acid substitution of the classical plasmid mediated β-lactamases like TEM-1, TEM- 2 and SHV-1 increases the spectrum of activity from earlier generation β-lactams to 3rd generation cephalosporins and monobactams 7. [ However, they retain their stability against cephamycins and carbapenems and are inhibited to an extent by β-lactamase inhibitors 6. Being plasmid mediated these enzymes spread fast amongst various bacteria and are important by infection control, clinical and therapeutic implication 8. The chromosomally mediated β-lactamase production is mainly through expression of AmpC gene which is either constitutive or inducible 5,9. Detection of organisms producing ESBLs provides helpful information to the clinicians. Treatment with newer cephalosporins or aztreonam may still result in treatment failure even when the causative organism appears to be susceptible to these antimicrobial agents by routine susceptibility testing 10. Many clinical laboratories currently test E. coli and Klebsiella spp. for ESBL production. However, there is scarcity of information on these isolates in United Arab Emirates (UAE). This study was conducted to: estimate the frequency of ESBLs in Gramnegative bacilli, detect different mechanisms of β lactam resistance (ESBLs, AmpC beta lactamases) and suggest a simple susceptibility testing protocol for an early presumptive identification of these enzymes in our laboratory. MATERIALS AND METHODS The study was conducted by the Department of Microbiology, Gulf Medical University (GMU), Ajman, United Arab Emirates from January 2010 to June 2010. The clinical isolates were obtained both from inpatients and outpatients attending Gulf Medical College Hospital, the teaching hospital of GMU. One hundred and twenty three isolates of Gram-negative bacilli were recovered from different clinical specimens submitted for routine microbiological analysis. This comprised of E.coli: 75, Klebsiella spp: 25, Pseudomonas aeruginosa: 12, Enterobacter spp.: 8, Citrobacter spp.: 2 and Proteus mirabilis: 1. Of the 123 isolates, 71 were cultured from GMJ. 2015;4(1):14-21 urine alone. Other clinical samples included pus/ wound aspirates, sputum, high vaginal swab, blood, and eye and ear secretions. The organisms were identified by standard identification techniques 11. Confirmation to the species level was done by API 20 E. Antimicrobial susceptibility of all the isolates was performed for amikacin, amoxicillin-clavulanic acid, cefotaxime, ceftriaxone, cefepime, ceftazidime, ceftazidime- clavulanic acid, cefoxitin, imipenem and aztreonam on Mueller Hinton Agar (MHA) using Modified Kirby-Bauer disk diffusion method as per Clinical Laboratory Standards Institute (CLSI) guidelines 12. The culture media and antibiotic disks were purchased from Hi-Media, India. E.coli ATCC 25922 and K.pneumoniae ATCC 700603 were used as controls to validate these susceptibility tests. Initial Screening Test: Isolates that exhibited resistance to any of the third generation cephalosporins (3GC) were the candidates for ESBL detection. An isolate was suspected to be an ESBL producer if it had the zones of inhibition for cefotaxime (30 μg) 27mm, ceftazidime (30 μg) 22mm, ceftriaxone (30 μg) 25mm and aztreonam (30 μg) 27mm 12. Further ESBL detection was carried out by two procedures: 1. Double Disk Synergy Test (DDST) 13 : The DDST was performed as a standard disk diffusion assay on MHA. Disks containing 30μg of ceftazidime and cefotaxime, each were placed 20mm apart (centre to centre) around a disk containing amoxicillin-clavulanic acid (20μg + 10μg). The MHA plate was incubated at 37 C overnight. Enhancement of inhibition zone of any one of the test antibiotics towards amoxicillin-clavulanic acid disk was regarded as presumptive ESBL production and subjected to phenotypic confirmatory test. [Figure]. 15

GMJ. 2015;4(1):14-21 ESBLS IN GRAM NEGATIVE BACILLI. were kept 15 mm apart from each other (center to center). Imipenem, an inducer, was placed in the centre and on either side of it, at a 15 mm distance, were placed ceftazidime and cefotaxime (indicators of induction). In addition, another inducer cefoxitin was placed at 15 mm from cefotaxime (indicator). This was placed opposite to that of ceftazidime -clavulanic acid to avoid any effect of inducible β-lactamase on the zone of inhibition of the latter. The remaining disks were placed as shown in figure 3. Figure 1: Enhanced zone of inhibition for ceftazidime and cefotaxime on the side of Amoxy-clav indicate ESBL production 2. Phenotypic confirmatory test for ESBL production: This test was performed on MHA by disk-diffusion method as recommended by CLSI. [12] A > 5 mm increase in zone diameter for ceftazidime tested in combination with clavulanic acid versus its zone when tested alone confirms an ESBL producing organism. [Figure 2] Figure 3: Novel method of placement of antibiotic disc to assess ESBL and AmpC production. Blunting of zone towards *Inducer indicates inducible AmpC production. Imipenem 1, Cefotaxime 2, Cefoxitin 3, Ceftazidime 4, Ceftazidime- Clavulanic Acid 5, Aztreonam 6, Ceftriaxone 7. The following criteria were used to categorize isolates exhibiting different mechanisms of resistance 5,12,14 : Figure 2: Phenotypic confirmatory test: A > 5 mm increase in zone diameter for ceftazidime in combination with clavulanic acid versus its zone when tested alone confirmed an ESBL producing organism. The novel method by Rodrigues et al to assess ESBL and other mechanisms of production of β-lactam resistance was also studied for each isolate 5,14. The ceftazidime and ceftazidime-clavulanic acid disks ESBL producer: i) zone diameters for various 3rd generation cephalosporins as mentioned above ii) susceptible to cefoxitin iii) increase in zone size with addition of an inhibitor by >5 mm Inducible AmpC producer: i) blunting of zone towards inducer ii) no increase in zone size with addition of an inhibitor iii) susceptible to cefepime 16

EKTA CHOURASIA, ET AL Derepressed mutants: i) resistant to cefoxitin and cefotaxime ii) no increase in zone size with addition of an inhibitor Multiple mechanisms: i) resistant to cefoxitin ii) blunting of zone towards inducer iii) increase in zone size with addition of an inhibitor by >5 mm GMJ. 2015;4(1):14-21 RESULTS We observed that ceftazidime was the most effective 3GC for initial screening of ESBL producers. Resistance to aztreonam was seen in 80% of the ESBL producing isolates. (Table 1) ESBL producing strains of Citrobacter spp. and Proteus mirabilis showed resistance to all 3GC. Table 1: Screening Test: Resistance of ESBL a producing Gram- negative bacilli to 3GC b and aztreonam ESBL positive bacterial strains No. (n) Aztreonam Cefotaxime Ceftazidime Ceftriaxone E.coli 29 24 (82.8%) 25 (86.2%) 28 (96.5%) 20 (69%) Klebsiella 10 6 (60%) 7 (70%) 10 (100%) 6 (60%) Pseudomonas 4 4 (100%) 3 (75%) 4 (100%) 3 (75%) Enterobacter 3 2 (66.7%) 3 (100%) 3 (100%) 2 (66.7%) a ESBL: extended spectrum beta-lactamase b 3GC 3 rd generation cephalosporins ESBL strains which tested positive by DDST were also found to be positive by the phenotypic confirmatory test. Of the 123 isolates, 48 (39.1%) were ESBL producers, of which 29 (60.4%) were E.coli, 10 (20.9%) Klebsiella spp., and 4 (8.3%) Pseudomonas aeruginosa. The commonest ESBL producers were E.coli and Klebsiella spp. (Figure 4) but the most frequent ESBL producer was Klebsiella spp. (40%) closely followed by E.coli (38.7%), Enterobacter spp. (37.5%) and Pseudomonas aeruginosa (33.3%). The novel method by Rodrigues et al showed that out of 123 isolates, 48 (39.1%) were ESBL producers of which 36 (29.3%) exhibited multiple mechanisms of resistance while remaining 12 (9.8%) were plain ESBL producers. Nine (7.3%) were plain derepressed mutants. Inducible AmpC β-lactamase production was detected only in Pseudomonas aeruginosa. Majority of ESBL producers and derepressed mutants belonged to Enterobacteriaceace. The percentage of different mechanism of β lactam resistance is shown in Table 2. Figure 4: Percentage of Common ESBL producers 17

GMJ. 2015;4(1):14-21 ESBLS IN GRAM NEGATIVE BACILLI. Table 2: Percentage of different mechanism of β lactam resistance Organism (Isolates) No. of isolates tested (n) Plain ESBL Multiple Mechanism Derepressed E.coli 75 7 (9.3%) 22 (29.3%) 4 (5.3%) - Klebsiella 25 3 (12%) 7 (28 %) - - Inducible Pseudomonas 12 1 (8.3%) 3 (25%) 2 (16.7%) 4 (33.3%) No resistance 42 (56.1%) 15 (60%) 10 (16.7%) Enterobacter 8-3 (37.5%) 3 (37.5%) - 2 (25%) Citrobacter 2-1 (50%) - - 1 (50%) Proteus 1 1 (100%) - - - - Total 123 12 (9.8%) 36 (29.3%) 9 (7.3%) 4 (3.3%) 62 (50.3%) Forty one percent of ESBL producers were isolated from urine whereas 22.3% were isolated from exudates. All ESBL producers and derepressed mutants were sensitive to imipenem. Among non β lactam antibiotics, 12% of ESBL producing isolates showed resistance to amikacin. DISCUSSION This study demonstrates the presence of ESBLmediated resistance in Gram-negative bacilli causing infections in patients attending our teaching hospital in Ajman, UAE. There have been numerous reports of ESBLs from different parts of the world but limited data is available from UAE 15. ESBL detection is not commonly carried out in many microbiology units in developing countries and this could be due to lack of awareness or lack of resources and facilities to conduct ESBL identification or both 16. The high rate of resistance amongst the isolates in our study is a matter of serious concern. Forty eight out of the 123 (39.1%) Gram-negative bacilli were ESBL producers. The prevalence of ESBL producers varies across continents and countries and also within hospitals. A predominance of either Klebsiella pneumoniae or E. coli has often been reported among the ESBL isolates identified worldwide 2-5,9,14-17. Previous studies from India have reported ESBL production varying from 6.6% to 91% 3,5,14,16,17. Studies in Pakistan show a variable rate ranging from 24% to 76% 2,18,19. A survey of intensive care units in Europe has found that the prevalence of ESBL in Klebsiella spp. varied from 3% in Sweden to 34% in Portugal 20. Higher figures of 30 60% have been reported from the South American countries of Brazil, Venezuela and Colombia 21-23. In Korea, Japan, Malaysia and Singapore, National Surveys have demonstrated the presence of ESBLs in 5 8% of E. coli isolates whereas higher rates of up to 24% has been reported from other Asian countries 10,24. ESBL production in Klebsiella spp. have also been reported to be as low as 5% in Japan and Australia with higher rates of 20 50% in other parts of the continent 10,24. In UAE, 41% of E.coli and Klebsiella spp. isolated over 14 months were found to be ESBL producers 15. In the Arabian Gulf region, 7.5% of Enterobacteriaceae and Pseudomonas spp. isolated over a one-year period in Kuwait were reported as being ESBL producers using the VITEK 2 system 25 whereas 23.6% of gram-negative bacilli were positive for ESBL production in Iran 26. In our study, Klebsiella spp. was the most frequent ESBL producer followed by E.coli which is in concordance with three other studies 15, 25, 27. E.coli was found to be the most frequent ESBL producer followed by Klebsiella spp. in studies conducted in India 5, 14 whereas Enterobacter cloacae (79%) was the most frequent ESBL producer in another study conducted in Pakistan. A higher rate of ESBL production by Klebsiella pneumoniae (55%) was observed in Riyadh 28. The novel method by Rodrigues et al indicated that ESBL producing organisms like Klebsiella spp., E.coli, Pseudomonas aeruginosa and Enterobacter spp. showed different mechanisms for 18

EKTA CHOURASIA, ET AL the production of multiple β-lactamases. Similar observations were made in other studies 5,14 The percentage of plain ESBL producers among E. coli (9.3%) and Klebsiella spp. (12%) was less frequent when compared to studies conducted by Shobha et al 14 and Varaiya et al 29 but the results have been comparable to the study done by Rodrigues et al 5. In this study, only 5.3% of E. coli and 16.7% of Pseudomonas spp. were derepressed mutants while this percentage was much higher in studies conducted by Rodrigues et al, and Shobha et al. 5,14 Pseudomonas aeruginosa was the only Gram-negative bacilli that produced inducible AmpC β-lactamases. One-third (33.3%) of Pseudomonas isolates showed inducible AmpC β-lactamase production while 26.5% of Pseudomonas spp. were inducible AmpC β-lactamase producer in a study done at Mumbai 5. When the phenotypic confirmatory method was compared to the novel method described by Rodrigues et al, the latter was found to be better than the former, because it assesses ESBL producers, de-repressed mutants, inducible AmpC β-lactamase production and multiple mechanisms in a single culture plate and it was also easy to perform. Although CLSI also recommends MIC broth microdilution for ESBL detection, we used disk diffusion method as it is comparatively simple and also cost effective. Similar to other reports, 15,30 the majority of Enterobacteriaceae isolates were from urine specimens, indicating the need for active screening of urine cultures for ESBL producers. Indeed, the detection of EBSL producers in urine has been described as representing an epidemiologic marker of colonization and potential for transfer between patients 10. Clinical isolates obtained from other sources were also found to be ESBL producers; hence it is imperative that active screening of Enterobacteriaceae isolates from all specimen types should be adopted to address the high prevalence rate of ESBL isolates. Unfortunately, many clinical microbiological laboratories still face significant problems with ESBL screening and identification as ESBL pathogens can present with variations in the in-vitro pattern of resistance to β-lactam agents 15. Enzymes such as TEM-3 and SHV-2 confer high levels of resistance to cephalosporins, whereas others, namely TEM-7 and TEM-12, confer low levels of resistance, which possibly makes detection of resistance even more difficult with the routinely used antimicrobial susceptibility tests 4,7, 8. GMJ. 2015;4(1):14-21 The antimicrobial susceptibility test showed that all the isolates including ESBL strains were susceptible to imipenem but 12% of the ESBL producers showed resistance to amikacin. Similar studies conducted in UAE and India 5,14,15 showed 100% susceptibility to amikacin and imipenem. Although amikacin and carbapenems are used only as the reserve drugs for treatment of infection by ESBL producing strains, production of β-lactamases capable of hydrolyzing carbapenems has also been reported mostly in Enterobacter spp. and Serratia spp 15. Carbapenem resistance is due to the production of chromosomal and plasmid-mediated cephaloporinases along with decreased permeability of drug through outer membrane. ESBL producers often show cross-resistance with non- β -lactam antibiotics, such as quinolones and aminoglycosides, resulting in limitation of antimicrobial choice. This co-resistance arises probably because these plasmidmediated enzymes are transferable between bacterial species and are also capable of incorporating genetic material coding for resistance to other antibiotics 4,8. While this Study was aimed at determining the prevalence of ESBL isolates in our hospital, the need for the molecular characterization of these isolates cannot be neglected. Although we could not carry out the molecular characterization due to lack of facilities and infrastructure, the finding of such resistant isolates in our setting is a matter of concern. CONCLUSION Clinical laboratories must be aware of the importance of ESBL and plasmid mediated AmpC β-lactamase production. Although CLSI recommendations exist, they are limited to ESBL producers of E. coli, Klebsiella spp. and Proteus mirabilis. No recommendations exist for ESBL detection and reporting for other organisms, or for the detection of AmpC β-lactamases. Screening methods of ESBL with recommended zone size should be immediately applied to suggest the presence of an ESBL. Large surveys, continued surveillance by clinical microbiology laboratory, judicious use of antimicrobial agents as well as implementation of infection control measures are recommended if the frequency of ESBL isolates is to be reduced in this setting. ACKNOWLEDGEMENT This work was supported by a grant from the 19

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